The overall objective of this proposal is to establish the role of connective tissue in tumor growth and metastasis. Recent evidence suggests that collagen synthesis by fibroblasts is transformation sensitive. Since epithelial cell carcinomas are more common in humans, our first specific aim is to examine collagen synthesis by chemically transformed epithelial cells. Our previous evidence showed that the established rat liver epithelial cell line, K16, produced type I collagen. However, the chemically transformed cell, W8, has lost the ability to synthesize the alpha 2(I) chain because the mRNA for this chain is absent. This year we will establish the reason for the absent mRNA by first using Southern blot analysis on isolated DNA from the W8 cells. This should establish whether the gene is deleted or altered. The RNA from nuclei will be isolated and analyzed by dot blots and/or Northern blots to determine if the initial transcript is not processed. Finally, if these techniques do not yield answers, we will attempt to establish a transcriptional assay system. Using similar technology, we will determine if mRNA for collagen decreases in M cells that were derived from W8 cells and have decreased collagen production and increased tumorigenicity. We will also determine if collagen synthesis is temperature sensitive in a cell line that is temperature sensitive for growth in soft agar. Our second aim is to determine if the tumor cell or stroma cell is responsible for collagen production in tumors. We have established that two guinea pig hepatocarcinoma cell lines, ln 1 and ln 10, produce type IV collagen in vitro, whereas guinea pig fibroblasts produce other types of collagen. This year we plan to localize the collagen types in the tumors, using immunofluorescence techniques. The ln 1 cells form collagenous tumors that regress whereas the ln 10 cells form invasive tumors with small amounts of collagen. This system will be used to determine if tumor products from ln 1 cells induce collagen deposition in the tumors and if this is responsible for the lack of invasiveness of the ln 1 tumors.